This project aims to define how changes in the composition of Haemophilus influenzae lipooligosaccharides affect biofilms. H. influenzae biofilms cause chronic otitis media infections that are among the most common and costly public health problems in the world. Biofilms formed in the middle ear during otitis media resist pharmaceutical or immune clearance. We recently discovered that the lipooligosaccharide glycolipids in H. influenzae biofilms contain sialic acid and phosphorylcholine that contribute to the progression of otitis in a chinchilla middle-ear infection model system. We hypothesize that changes in the composition of H. influenzae lipooligosaccharides promote the formation of mature biofilms, thus increasing resistance to clearance, and decreasing innate responses. To address this hypothesis, we will complete the following Specific Aims: Specific Aim 1: To delineate how lipooligosaccharide composition promotes H. influenzae biofilm phenotypes in vitro. Benchmarks for defining mature biofilms include decreased host inflammatory responses, and increased biofilm density and structure that promotes resistance to killing by antibiotics. Specific Aim 2: To establish the role of biofilm-related changes in lipooligosaccharides in H. influenzae otitis media. We will use the chinchilla animal model for otitis media to evaluate how changes in lipooligosaccharide composition observed in biofilms in vitro increase H. influenzae persistence and virulence in vivo. The completion of these Specific Aims will elucidate biofilm growth mechanisms that are critical to otitis media and other chronic infections. No vaccine in current use protects against the unencapsulated H. influenzae strains that cause otitis, and antibiotic resistance increasingly limits treatment options for these infections. This problem is made even worse by the inherent resistance of biofilms to most antibiotics. A better understanding of biofilms will be crucial to the rational design of new ways to diagnose, treat or prevent chronic otitis media. The research proposed here thus presents an important opportunity to identify new targets for eliminating one of the most costly and prevalent problems in public health worldwide.